The development of rapid genotyping methods for methicillin-resistant Staphylococcus aureus

Methicillin-resistant Staphylococcus aureus (MRSA) is an important human pathogen that is endemic in hospitals all over the world. It has more recently emerged as a serious threat to the general public in the form of community-acquired MRSA. MRSA has been implicated in a wide variety of diseases, ranging from skin infections and food poisoning to more severe and potentially fatal conditions, including; endocarditis, septicaemia and necrotising pneumonia. Treatment of MRSA disease is complicated and can be unsuccessful due to the bacterium's remarkable ability to develop antibiotic resistance. The considerable economic and public health burden imposed by MRSA has fuelled attempts by researchers to understand the evolution of virulent and antibiotic resistant strains and thereby improve epidemiological management strategies. Central to MRSA transmission management strategies is the implementation of active surveillance programs, via which unique genetic fingerprints, or genotypes, of each strain can be identified. Despite numerous advances in MRSA genotyping methodology, there remains a need for a rapid, reproducible, cost-effective method that is capable of producing a high level of genotype discrimination, whilst being suitable for high throughput use. Consequently, the fundamental aim of this thesis was to develop a novel MRSA genotyping strategy incorporating these benefits. This thesis explored the possibility that the development of more efficient genotyping strategies could be achieved through careful identification, and then simple interrogation, of multiple, unlinked DNA loci that exhibit progressively increasing mutation rates. The baseline component of the MRSA genotyping strategy described in this thesis is the allele-specific real-time PCR interrogation of slowly evolving core single nucleotide polymorphisms (SNPs). The genotyping SNP set was identified previously from the Multi-locus sequence typing (MLST) sequence database using an in-house software package named Minimum SNPs. As discussed in Chapter Three, the genotyping utility of the SNP set was validated on 107 diverse Australian MRSA isolates, which were largely clustered into groups of related strains as defined by MLST. To increase the resolution of the SNP genotyping method, a selection of binary virulence genes and antimicrobial resistance plasmids were tested that were successful at sub typing the SNP groups. A comprehensive MRSA genotyping strategy requires characterisation of the clonal background as well as interrogation of the hypervariable Staphylococcal Cassette Chromosome mec (SCCmec) that carries the β-lactam resistance gene, mecA. SCCmec genotyping defines the MRSA lineages; however, current SCCmec genotyping methods have struggled to handle the increasing number of SCCmec elements resulting from a recent explosion of comparative genomic analyses. Chapter Four of this thesis collates the known SCCmec binary marker diversity and demonstrates the ability of Minimum SNPs to identify systematically a minimal set of binary markers capable of generating maximum genotyping resolution. A number of binary targets were identified that indeed permit high resolution genotyping of the SCCmec element. Furthermore, the SCCmec genotyping targets are amenable for combinatorial use with the MLST genotyping SNPs and therefore are suitable as the second component of the MRSA genotyping strategy. To increase genotyping resolution of the slowly evolving MLST SNPs and the SCCmec binary markers, the analysis of a hypervariable repeat region was required. Sequence analysis of the Staphylococcal protein A (spa) repeat region has been conducted frequently with great success. Chapter Five describes the characterisation of the tandem repeats in the spa gene using real-time PCR and high resolution melting (HRM) analysis. Since the melting rate and precise point of dissociation of double stranded DNA is dependent on the size and sequence of the PCR amplicon, the HRM method was used successfully to identify 20 of 22 spa sequence types, without the need for DNA sequencing. The accumulation of comparative genomic information has allowed the systematic identification of key MRSA genomic polymorphisms to genotype MRSA efficiently. If implemented in its entirety, the strategy described in this thesis would produce efficient and deep-rooted genotypes. For example, an unknown MRSA isolate would be positioned within the MLST defined population structure, categorised based on its SCCmec lineage, then subtyped based on the polymorphic spa repeat region. Overall, by combining the genotyping methods described here, an integrated and novel MRSA genotyping strategy results that is efficacious for both long and short term investigations. Furthermore, an additional benefit is that each component can be performed easily and cost-effectively on a standard real-time PCR platform

New Keck Observations of Lithium in Very Metal-poor Stars

Lithium abundances have been determined in more than 100 metal-poor halo stars both in the field and in clusters. From these data we find trends of Li with both temperature and metallicity and a real dispersion in Li abundances in the Spite Li plateau. We attribute this dispersion primarily to Li depletion (presumably due to extra mixing induced by stellar rotation) and to Galactic chemical evolution. We derive a primordial Li of 2.44 $\pm$0.18 for A(Li)$_p$ = log N(Li/H) + 12.00. This agrees with the Li abundances predicted by the $WMAP$ results. For stars cooler than the Li plateau we have evidence that Li depletion sets in at hotter temperatures for the higher metallicity stars than for the low-metal stars. This is the opposite sense of predictions from stellar models. The smooth transition of the Li content from the Li plateau stars to the cool stars adds weight to the inference of Li depletion in the plateau stars.Comment: Invited talk for IAU Symposium 228 "From Lithium to Uranium..." held in Paris in May, 2005. 6 pages, 6 figure

Primordial Lithium: Keck Observations in M92 Turnoff Stars

We present new Keck I/HIRES observations at R\45,000 (\3 pixels) of seven stars near the turno† of the old, metal-poor globular cluster M92. In three of these stars, we have signal-to-noise ratios (S/Ns) of 40 pixel~1, and in the other four, the S/N is near 20. The Li abundance in star 18 is high compared with the halo Ðeld-star plateau and is similar to that in the remarkable Li-rich halo Ðeld star BD ]23¡3912. In addition to the high Li abundance in star 18, there is a dispersion in Li abundance in our seven stars covering the full range of a factor of 3. We have attempted to determine whether the excess Li in star 18 is due to less than average Li deple-tion in this star from an even higher initial abundance, as predicted by the Yale rotational models, or whether it is due to the extraordinary action of Li production mechanisms in the material that formed this star. We have found no convincing evidence that favors Li production: (1) Stars 18, 21, and 46 have identical Ba abundances, which argues against Li production carrying an s-process signature. (2) These three stars have indistinguishable Ca, Cr, Fe, and Ti, which argues against supernova Li production. (3) We discuss l-process production of Li and Ðnd no convincing observational evidence for this from the strengths of the Mg, Ca, and Fe lines. (4) The similarity in age of these cluster stars argues against cosmic-ray Li production that requires age di†erences of gigayears. The most likely explanation for the Li dispersion is di†erential Li depletion from a (possibly signiÐcantly) higher primordial Li abundance due to di†erences in the initial angular momentum in each star followed by spin-down; the most rapid rotators destroy the most Li, whereas the initially slower rotators preserve more Li

Lithium and Lithium Depletion in Halo Stars on Extreme Orbits

We have determined Li abundances in 55 metal-poor (3.6 < [Fe/H] < -0.7) stars with extreme orbital kinematics. We find the Li abundance in the Li-plateau stars and examine its decrease in low-temperature, low-mass stars. The Li observations are primarily from the Keck I telescope with HIRES (spectral resolution of ~48,000 and median signal-to-noise per pixel of 140). Abundances or upper limits were determined for Li for all the stars with typical errors of 0.06 dex. Our 14 stars on the Li plateau give A(Li) = log N(Li)/N(H) + 12.00 of 2.215 +-0.110, consistent with earlier results. We find a dependence of the Li abundance on metallicity as measured by [Fe/H] and the Fe-peak elements [Cr/H] and [Ni/H], with a slope of ~0.18. We also find dependences of A(Li) with the alpha elements, Mg, Ca, and Ti. For the n-capture element, Ba, the relation between A(Li) and [Ba/H] has a shallower slope of 0.13; over a range of 2.6 dex in [Ba/H], the Li abundance spans only a factor of two. We examined the possible trends of A(Li) with the characteristics of the orbits of our halo stars, but find no relationship with kinematic or dynamic properties. The stars cooler than the Li plateau are separated into three metallicity subsets. The decrease in A(Li) sets in at hotter temperatures at high metallicities than at low metallicities; this is in the opposite sense of the predictions for Li depletion from standard and non-standard models.Comment: 29 pages including 3 tables and 12 figures Accepted by The Astrophysical Journal, for the 1 November 2005 issue, v. 63

Keck HIRES Spectroscopy of M92 Subgiants: Surprising Abundances Near the Turnoff

Using high-resolution, moderate signal-to-noise ratio spectroscopy obtained with the 10 m Keck I Telescope and efficient HIRES echelle spectrograph, we derive abundances of several elements in subgiants near the M92 turnoff. As a consistency check, we also analyze the metal-poor field star HD 140283 and find an Fe abundance in fine agreement with many previous determinations. However, our M92 value ([Fe/H] = -2.52) is a factor of 2 lower than the abundance derived from its red giant members. Differences in model atmospheres, gf-values, and instrumental effects might account for this difference, but whether they in fact do so is unclear. We note possible evidence for [Fe/H] differences within M92. Our spectroscopic analysis suggests that the M92 reddening, E(B-V), may be 0.04–0.05 mag greater than canonical values, but various uncertainties mean that this conclusion is not definitive; the significant difference in interstellar Na I line strengths in the M92 and HD 140283 spectra may be consistent with an increased reddening. Regardless, the conclusion that either the [Fe/H] of M92 has been significantly overestimated from red giants or current reddening/photometry estimates are too small/red is not easily escaped. If the reddening/photometry were in error by this amount, turnoff color–based ages for M92 could be reduced by ~4 Gyr. The adjustment to the M92 distance modulus required for a similarly reduced turnoff age that is luminosity-based can be accommodated by increases in extinction and alterations to the metal-poor field star distance scale recently inferred from Hipparcos Cepheid and subdwarf data

Beryllium in Lithium-Deficient F and G Stars

We present the results of an extensive search, conducted at the Canada-France-Hawaii 3.6-m tele-scope, for beryllium (Be) in the atmospheres of lithium-deÐcient F and G dwarfs. We also report revised lithium (Li) estimates for the entire sample using previously published equivalent widths and updated, consistently calculated stellar parameters. Abundances derived from an LTE analysis of the Li and Be line-forming regions conÐrm the suspicion that F stars which deplete Li by factors of 10È200 may also be beryllium deÐcient. Photospheric Be concentrations range from near meteoritic levels in G dwarfs to factors of 10È100 below this assumed initial abundance in hotter stars. Moreover, signiÐcant Be deÐ-ciencies appear in stars that populate a 600 K wide e†ective temperature window centered on 6500 K. This Be abundance gap is reminiscent of the Li gap observed in open clusters. Also, the discovery of 12 probable ““110 HerculisÏÏ stars, objects that exhibit a depleted, but detected, surface concentration of both Li and Be, provides a powerful means of di†erentiating between the possible physical processes responsible for observed light element abundance patterns. Indeed, the Be data presented here, in con-junction with the newly calculated Li abundances, lead to the following conclusions regarding the hypothesized, light element depletion scenarios: Mass loss cannot account for stars with severely depleted (but detected) Li and moderate Be deÐciencies. The predicted timescales for surface depletion due to microscopic di†usion are too long for signiÐcant Li and Be deÐciencies to develop in cool (T¹ 6200) stars; nevertheless, underabundances are observed in these stars. Di†usion theory also predicts Li and Be depletion rates to be comparable, but it is evident that Li and Be depletion proceed at di†erent speeds. Models of mixing induced by internal gravity waves cannot explain mild Be deÐciencies in cool dwarfs. A key meridional circulation prediction regarding the efficiency and severity of Li and Be dilu-tion is shown to be fallible. However, rotationally induced mixing, a turbulent blending of material beneath the surface convection zone due to the onset of instabilities from superÐcial angular momentum loss, predicts both the observed light element depletion morphology as well as the existence of 110 Her analogs. These ““YaleÏÏ mixing models provide, therefore, the most plausible explanation, of those presented, for the observed Li and Be abundances

Defining Identity Crimes

The objective of this paper is to report on the definitions of the terms used and in use across different regions for identity crime, namely, identity fraud, identity theft, and identity deception. The purpose is to clarify the meaning of the terms used with a view to gaining a consensus amongst the various stakeholders. This consensus is essential to enable further research. Without consensus measurement and comparisons are meaningless. Our study of identity fraud has an industry-driven research agenda. A grounded theory research methodology is used when interviewing government agencies and private organisation participants. Interviews sought to better understand current information and communications technology (ICT) practitioners’ security and privacy issues with respect to identity fraud perpetrator attacks. We found there to be consensus among stakeholders for the meaning of identity fraud and identity theft but less agreement for identity deception

The molybdenum isotopic compositions of I-, S- and A- type granitic suites

JY is funded by a Clarendon Scholarship and a Dr Bill Willetts Scholarship from the University of Oxford. The research leading to these results has received funding from the European Research Council under the European Union's Seventh Framework Programme (FP7/2007-2013) / ERC grant agreement No. 247422. This work has also supported by Science & Technology Facilities Council.This study reports Mo isotopic compositions for fifty-two Palaeozoic granitic rocks with contrasting source affinities (A-, I- and S-type) from the Lachlan Fold Belt (LFB) and the New England Batholith (NEB), both in SE Australia, and three compositionally zoned plutons (Loch Doon, Criffell, and Fleet) located in the South Uplands of Scotland. The results show relatively large variations in δ98Mo for igneous rocks ranging from -1.73‰ to 0.59‰ with significant overlaps between different types. No relationships between δ98Mo and δ18O or ASI (Alumina Saturation Index) are observed, indicating that Mo isotopes do not clearly distinguish igneous versus sedimentary source types. Instead, effects of igneous processes, source mixing, regional geology, as well as hydrothermal activity control the Mo isotope compositions in these granites. It is found that Mo is mainly accommodated in biotite and to a lesser extent in hornblende. Hornblende and Fe3+-rich minerals may preferentially incorporate light isotopes, as reflected by negative correlations between δ98Mo and K/Rb and [Fe2O3]. There is a positive correlation between initial 87Sr/86Sr and δ98Mo in I-type granitic rocks, reflecting the admixing of material from isotopically distinct sources. Granitic rocks from Scotland and Australia display strikingly similar curvilinear trends in δ98Mo vs. initial 87Sr/86Sr despite the differing regional geology. Localized hydrothermal effects on Mo isotopes in three samples from Loch Doon and Criffell can result in anomalously low δ98Mo of < -1‰. Based on this study, an estimate of δ98Mo = 0.14±0.07‰ (95% s.e.) for the Phanerozoic upper crust is proposed. This is slightly heavier than basalts indicating an isotopically light lower crust and / or a systematic change to the crust resulting from subduction of isotopically light dehydrated slab and / or pelagic sediment over time.Publisher PDFPeer reviewe